Therapeutic efficacy of a novel immunotherapeutic formulation, combining a multi-epitope chimeric protein and Amphotericin B, in murine L. infantum infection.

Leishmaniasis is a vector-borne parasitic disease caused by Leishmania protozoa. First-line treatment with pentavalent antimonials faces increasing resistance and significant toxicity. Amphotericin B is an effective second-line alternative but is also toxic and complex to administer, with a safer but expensive liposomal formulation. This study evaluated an immunotherapeutic approach using a chimeric protein composed of multiple T-cell epitopes from Leishmania (L.) infantum, combined with the adjuvant Monophosphoryl Lipid A (MPLA), in a murine model of VL. Our findings demonstrate that the combined rChi+MPLA/Amp-B therapy significantly outperformed monotherapies, inducing a robust TH1-polarized immune response. Splenocytes from this group exhibited markedly higher levels of TNF in both CD4+ (SC/CC = 1.64) and CD8+ T cells (SC/CC = 1.93) compared to Saline, MPLA, and rChi controls (p < 0.05). Furthermore, the combination therapy achieved a significantly higher IFN-γ/IL-10 ratio in CD4+ cells (Ratio = 2.03) compared to Saline, MPLA, and rChi groups (p < 0.05), indicating a shift away from the immunosuppressive environment characterized by high IL-10 levels in untreated Saline controls (SC/CC = 1.58). This protective environment was further corroborated by a marked elevation in nitric oxide (NO) levels in the rChi+MPLA/Amp-B group compared to all other experimental groups (p < 0.05). This potent immune activation correlated with a drastic reduction in parasite load. The rChi+MPLA/Amp-B group achieved statistically significant reductions (p < 0.05) of 98.4% in the lymph nodes, 99.1% in the spleen, and 97.8% in the liver compared to the Saline group. These reductions were also significantly superior (p < 0.05) to those observed in animals treated with Amp-B alone (82.1%, 89.3%, and 79.4%) or rChi+MPLA alone (75.2%, 68.5%, and 72.1%) in the lymph nodes, spleen, and liver, respectively. These results highlight the potential of the rChi+MPLA/Amp-B formulation as a highly effective immunotherapeutic strategy to enhance conventional chemotherapy and improve the clinical management of VL.